N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

Endpoint summary

Description of key information

The skin sensitisation endpoint has been fulfilled using a weight-of-evidence approach based on QSAR calculation, in vitro and in vivo data. 

The QSAR calculation results indicated that the substance does not possess chemical structures associated with skin sensitisation and hence a prediction of "not a skin sensitiser" was aligned to this substance.

In an in vitro skin sensitisation test conducted in accordance with OECD Guideline 442E, the results of this study indicates that the substance is not a skin sensitiser under conditions of the test.

In an in vivo skin sensitisation test (LLNA-ELISA type) conducted in accordance with OECD Guideline 442B, the mean SI values for the 3 test concentrations tested where all <3.  This indicates that the substance is not a skin sensitiser under conditions of the test.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Part of weight-of-evidence approach adapting the information requirements of Annex VII 8.3.1 and 8.3.2. under REACH in accordance with Annex XI Section 1.2. A sequential series of skin sensitisation tests were performed which collectively provide all the information required to satisfy the information endpoint for Annex VII 8.3.1. and 8.3.2 under REACH. Therefore in accordance with Annex XI, 1.2 of the REACH Regulation is no additional testing is scientifically necessary based on a weight-of evidence approach.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin sensitisation: Local Lymph Node Assay: BrdU-ELISA or –FCM)

Version / remarks:

Appendix IA, June 25 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Japan
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8 weeks for pre-screen, 9 weeks for main study
- Weight at study initiation: See Main study Day 1 - Table 3 in results sections
- Housing: polycarbonate cages with wood chips and environmental enrichment
- Diet (e.g. ad libitum): pelleted diet, ad libitum
- Water (e.g. ad libitum): chlorinated water, ad libitum
- Acclimation period: 7 days for pre-screen, 14 days for main study
- Indication of any skin lesions: all animals in good health condition

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

dimethylformamide

Concentration:

10%, 25% and 50%

No. of animals per dose:

4 animals per test item concentration
4 animals for vehicle control
4 animals for positive control

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Vehicle solubility trials were performed. Recommended vehicles in the OECD 442, acetone: olive oil (4:1 v/v/ AOO), DMF, methyl ethyl ketone (MEK) and dimethylsulfoxide (DMSO) were selected for solubility trials. The test item dissolved in DMF and DMSO at 50% (v/v), however, after 5 hours test item crystallised in the DMSO. The test item was not soluble on MEK or AOO. and therefore DMF selected as the vehicle for the pre-screen and main study.
- Irritation: none
- Systemic toxicity: none (no abnormalities reported)
- Ear thickness measurements: yes (see Table 2 in the results section)
- Erythema scores: no erythema so not scored

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item was regarded as a "sensitiser" when the SI of the test item was 2.0 or more and was regarded as "non-sensitiser" when the SI of the test item group was less than 1.6. When the SI was between 1.6 and 1.9, dose-response relationship and statistical significance would be considered.

TREATMENT PREPARATION AND ADMINISTRATION:
The test solution was prepared on each sensitisation day. 0.5 was dissolved in DMF and filled up to make 1mL of 50 w/v% solution. The 50 w/v% solution was serially diluted to prepare the test solutions at 25 and 10 w/v%.
25µL of each formulation was applied to the dorsum of each ear of the animals using a micropipette once per day for 3 consecutive days.
Approx. 48 hours after the final application of the formulations, 0.5 mL of BrdU solution was administrated intraperitoneally to each animal using a syringe and a needle. Approx. 24 hours after the BrdU administration, animals were humanely killed and each auricular lymph node was taken. The lymph nodes were carefully dissected and trimmed of surrounding tissue and fat, weighed both sides together. The mean values and standard deviations of the local lymph node weights were calculated for each group. The lymph nodes were stored individually in a biomedical freezer.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

No statistic performed as SI values all <1.6.

Positive control results:

SI = 2.93 +/-0.24.
The SI value was >2 and therefore indicates that the test system was functioning as intended.

Parameter:

SI

Value:

1.08

Variability:

S.E. +/- 0.12

Test group / Remarks:

10%

Remarks on result:

other: Mean SI value

Parameter:

SI

Value:

1.18

Variability:

S.E. +/- 0.15

Test group / Remarks:

25%

Remarks on result:

other: Mean SI value

Parameter:

SI

Value:

1.48

Variability:

S.E. +/- 0.21

Test group / Remarks:

50%

Remarks on result:

other: Mean SI value

Parameter:

SI

Value:

2.93

Variability:

S.E. +/- 0.24

Test group / Remarks:

Positive control

Remarks on result:

other: Mean SI value

Parameter:

SI

Value:

1

Variability:

S.E. +/- 0.04

Test group / Remarks:

Vehicle control

Remarks on result:

other: Mean SI value

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Lymph node weight - see Table 5

DETAILS ON STIMULATION INDEX CALCULATION


EC3 CALCULATION: not calculated

CLINICAL OBSERVATIONS:
See Table 4

BODY WEIGHTS
See Table 3

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness). None.

Table 1: Body weights in the pre-screen test

Exp. group		Animal No.	Body weights (g)
Group	Concentration (w/v%)		Day 1	Day 6^a)
Test item	10.0	1	22.1	22.2 (100.5)
	25.0	2	22.4	23.3 (104.0)
	50.0	3	22.4	23.1 (103.1)

Figures in parentheses indicate percentages compared to the initial body weight (Day 1)

 

Table 2: Thicknesses of auricle in the pre-screen test

Exp. Group		Animal No.	Thickness of auricle (mm)
Group	Concentration (w/v%)		Day 1		Day 3^a)		Day 6^a)
Test item			Left	Right	Left	Right	Left	Right
	10.0	1	0.185	0.190	0.205 (110.8)	0.210 (110.8)	0.190 (102.7)	0.205 (107.9)
	25.0	2	0.205	0.195	0.195 (95.1)	0.210 (107.7)	0.210 (102.4)	0.210 (107.7)
	50.0	3	0.180	0.190	0.200 (111.1)	0.210 (110.5)	0.200 (111.1)	0.195 (102.6)

Figures in parentheses indicate percentages compared to the initial thicknesses (Day 1)

 

Table 3: Body weights in the main study

Exp Group		Animal No.	Body weights (g)
Group	Concentration (w/v%)		Day 1		Day 6
			Individual	Mean ± S.D.	Individual	Mean ± S.D.
Vehicle Control (DMF)	-	1	22.3	22.43 ±1.80	22.8	23.15 ± 1.88
		2	25.0		25.9
		3	21.4		21.8
		4	21.0		22.1
Positive Control (HCA)	25.0	5	24.7	23.45 ± 2.43	24.4	23.35 ± 1.92
		6	26.2		25.4
		7	20.9		21.1
		8	22.0		22.5
Test item	10.0	9	24.5	22.90 ± 1.49	23.9	22.50 ± 1.27
		10	23.8		23.0
		11	21.9		20.9
		12	21.4		22.2
	25.0	13	22.2	22.55 ± 1.84	23.0	22.70 ± 1.22
		14	25.2		24.2
		15	21.0		21.3
		16	21.8		22.3
	50.0	17	22.5	22.45 ± 1.40	23.7	22.80 ± 1.34
		18	24.0		24.1
		19	20.6		21.2
		20	22.7		22.2

S.D: Standard deviation

DMF: N,N-dimethylformamide

HCA: α-hexylcinnamaldehyde

 

Table 4: Clinical signs in the main study

Exp Group		Animal No.	Observation period
Group	Concentration (w/v%)
			Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
Vehicle Control (DMF)	-	1	-	-	-	-	-	-
		2	-	-	-	-	-	-
		3	-	-	-	-	-	-
		4	-	-	-	-	-	-
Positive Control (HCA)	25.0	5	-	-	-	-	-	-
		6	-	-	-	-	-	-
		7	-	-	-	-	-	-
		8	-	-	-	-	-	-
Test item	10.0	9	-	-	-	-	-	-
		10	-	-	-	-	-	-
		11	-	-	-	-	-	-
		12	-	-	-	-	-	-
	25.0	13	-*	-*	-*	-	-	-
		14	-*	-*	-*	-	-	-
		15	-*	-*	-*	-	-	-
		16	-*	-*	-*	-	-	-
	50.0	17	-*	-*	-*	-	-	-
		18	-*	-*	-*	-	-	-
		19	-*	-*	-*	-	-	-
		20	-*	-*	-*	-	-	-

DMF: N,N-dimethylformamide

HCA: α-hexylcinnamaldehyde

- : no abnormalities detected

*-: test item crystallised on ear (after application)

 

Table 5: Lymph node weights in the main study

Exp Group		Animal No.	Lymph node weights (g)
Group	Concentration (w/v%)
			Individual	Mean ± S.D.
Vehicle Control (DMF)	-	1	4.3	4.28 ± 0.21
		2	4.5
		3	4.0
		4	4.3
Positive Control (HCA)	25.0	5	9.1	8.33 ± 0.80
		6	8.5
		7	7.2
		8	8.5
Test item	10.0	9	4.7	4.65 ± 0.34
		10	4.5
		11	4.3
		12	5.1
	25.0	13	4.7	4.35 ± 0.39
		14	4.4
		15	4.5
		16	3.8
	50.0	17	4.7	5.20 ± 0.36
		18	5.4
		19	5.2
		20	5.5

S.D: Standard deviation

DMF: N,N-dimethylformamide

HCA: α-hexylcinnamaldehyde

 

Table 6: BrdU labelling indices and stimulation indices in the main study

Exp Group		Animal No.	BrdU labelling index		Stimulation index
Group	Concentration (w/v%)
			Individual	Mean ± S.E.	Individual	Mean ± S.E.
Vehicle Control (DMF)	-	1	0.176	0.1560 ± 1.80	1.1	1.00 ± 0.04
		2	0.153		1.0
		3	0.136		0.9
		4	0.159		1.0
Positive Control (HCA)	25.0	5	0.568	0.4570 ± 0.0395	3.6	2.93 ± 0.24
		6	0.384		2.5
		7	0.426		2.7
		8	0.450		2.9
Test item	10.0	9	0.216	0.1708 ± 0.0169	1.4	1.08 ± 0.12
		10	0.143		0.9
		11	0.177		1.1
		12	0.147		0.9
	25.0	13	0.233	0.1835 ± 0.0215	1.5	1.18 ± 0.15
		14	0.185		1.2
		15	0.188		1.2
		16	0.128		0.8
	50.0	17	0.169	0.2285 ± 0.0322	1.1	1.48 ± 0.21
		18	0.243		1.6
		19	0.189		1.2
		20	0.313		2.0

S.E: Standard error

DMF: N,N-dimethylformamide

HCA: α-hexylcinnamaldehyde

Conclusions:

The SIs of the 50.0, 25.0 and 10.0 w/v% test item groups were 1.48, 1.18 and 1.08 (all SIs less than the cut-off value of 1.6. Therefore under the condition of the test, the test item was judged to be non-sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: weight of evidence

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: Test item was weighed and dissolved in DMSO using a laboratory mixer to prepare the test stock solution
- Preparation of the test chemical serial dilutions: Solutions prepared just for before use, stored at room temperature under yellow light and used within 2 hours. Diluted using DMSO and prepared under yellow light.
- Preparation of the positive controls: The positive control was weighed and dissolved in DMSO using a laboratory mixer to prepare the 100mg/mL solution. This solution was diluted with DMSO to prepare 2.0 mg/mL solution. Solution prepared just for before use, stored at room temperature under yellow light and used within 2 hours.
- Preparation of the solvent, vehicle and negative controls: not specified in detail. Negative control stored in freezer (-10 to -30°C )
- Stable dispersion obtained: yes

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 1000µg/mL (first test); 10µg/mL (second test)
- Solubility in solvents: yes
- Solubility in incubation medium: 62.5µg/mL or more, precipitation was observed (first test); non precipitation observed in test concentration (second test)
- Results of selecting appropriate concentration and determination of cytotoxicity: CV75
- Final concentration range selected on basis of the CV75 results. The highest dose was set at 3.83 µg/mL equivalent to 1.2 times CV75 and 7 doses set based on a geometric progression of 1.2.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates:
- Number of repetitions: 3 mains tests (3 confirmatory test)
- Test chemical concentrations: 1.07, 1.28, 1.54, 1.85. 2.22, 2.66, 3.19 and 3.83 µg/mL (main tests); 1.13, 1.36, 1.63, 1.96, 2.35, 2.82, 3.38, 3.55, 3.73, 3.91 and 4.11 µg/mL
- Application procedure: Each test solution was added to each well which cells were seeded and mixed then incubated. Non-treatment groups - 500µL of the medium was added.
- Exposure time: 24 +/- 0.5 hours
- Study evaluation and decision criteria used: Flow cytometry analysis; calculation of Relative Fluorescence Intensity (RFI). If the RFI of CD86 was 150 or more at any dose with cell viability >/=50%, and/or if the result of the RFI of CD54 was 200 or more at any dose with cell viability >/=50%, the result was considered positive. Otherwise it was judged to be negative.
- Description on study acceptance criteria: In the non-treatment group and the negative control group, it was judged negative for both CD86 and CD54 and cell viabilities were more than 90%. In the positive control group, it was judged positive for both CD86 and CD54 and cell viability was 50% or more. In the non-treatment group and the negative control group, the MFI ratios of both CD86 and CD54 to isotype were greater than 105%.
For the test item group, the cell viabilities were greater than 50% in at least four tested in each main test. When any criterion was not satisfied, the test results was rejected and the re-test of the test was carried out.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): <30; cell density was not to exceed 1x10E6 cells/mL during passage.
- Incubation conditions: CO2 incubator; 37°C; CO2 concentration 5%; under humid condition

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
- Flow cytometry used: Flow cytometer (Navios EX, Beckman Coulter)
- Plate used: 96-well round-bottom plate
- Preparation for CD54 and/or CD86 expression measurements/cell staining: the mixture of working solution and cell suspension after treatment were transferred into 2 mL sample tubes. The cell were collected by centrifugation. The supernanants were discarded and the remaining cells were suspended 1 mL of staining buffer. The cells were collected by centrifugation and then washed one with 1mL of staining buffer. The supernanants were discarded and the remaining cells were suspended in 600µL of globulin solution and incubated at 4°C for 15 minutes. Cells were divided into 3 aliquots of 180µL into 96-well round-bottom plate and were collected by centrifugation. The supernanents were discarded and the cells were stained with 50µL of each antibody solution at 4°C for 30 minutes in the dark.
- Propidium iodide staining/cytotoxicity measurements: yes; Cell viability was calculated by measuring a total of 10000 living cells (PI negative). 150µL of staining buffer was added to each well. The cells collected by centrifugation. The supernanants were discarded and remaining cells were suspended with 200µL . Cells collected by centrifugation and then washed one with 200µL of staining buffer. The supernanants were discarded and the remaining cells were suspended with 200µL of staining buffer. The cell suspension were transferred into tubes with 180µL staining buffer. 20µL of PI solution (12.5 µg/mL) was added to each tube (final concentration of PI = 0.625 µg/mL).

DATA EVALUATION
- Cytotoxicity assessment: The cell viability of each treatment group was exhibited by the cell viability when stained with isotype control

Vehicle / solvent control:

DMSO

Negative control:

other: Culture cell medium

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

The positive control was judged to be positive for both CD86 and CD54 and cell viability was 50% or more.

Group:

test chemical

Run / experiment:

other: 1st confirmatory test

Parameter:

RFI CD54>150 [442E]

Cell viability:

four or more doses were 50% or more viability

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The RFI values were below 150 when the cell viability was 50% or more in each dose.

Group:

test chemical

Run / experiment:

other: 1st confirmatory test

Parameter:

RFI CD86>200 [442E]

Cell viability:

four or more doses were 50% or more viability

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The RFI values were below 200 when the cell viability was 50% or more in each dose.

Group:

test chemical

Run / experiment:

other: re-run 2nd confirmatory test

Parameter:

RFI CD54>150 [442E]

Cell viability:

four or more doses were 50% or more viability

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The RFI values were below 150 when the cell viability was 50% or more in each dose.

Group:

test chemical

Run / experiment:

other: re-run 2nd confirmatory test

Parameter:

RFI CD86>200 [442E]

Cell viability:

four or more doses were 50% or more viability

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The RFI values were below 200 when the cell viability was 50% or more in each dose.

Other effects / acceptance of results:

6 experiments were run in total:
- 1st main test - negative - REJECTED
- 2nd main test - positive - REJECTED
- 3rd main test - negative - REJECTED
Negative results obtained in 2 out of 3 main tests. The RFIs of CD54 was 200 or more at 3.83 µg/mL at which the cell viabilities were approx. 30%. No doses had cell viabilities between 50 - 90%. In order to determine whether the RFI of CD54 increases at between 3.19 µg/mL and 3.83 µg/mL, the confirmation test was conducted, rejecting the results of the main tests.
- 1st confirmatory test - Negative - VALID (see Table 1)
- 2nd confirmatory test - REJECTED - RFIs were not dose-related for CD86 and CD54
- re-run 2nd confirmatory test - Negative - VALID (see Table 2)

Conclusions:

The item item was classified as negative by h-CLAT under the test conditions.

Reference 3

Endpoint:

skin sensitisation: in chemico

Remarks:

QSAR result

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
QSAR

2. MODEL (incl. version number)
DEREK NEXUS 6.1.0.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
c1cc(ccc1S(-O)(Oc2cc(ccc2)NC(=O)Nc3cc(ccc3)OS(=O)(c4ccc(cc4)C)=O)=O)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF.

5. APPLICABILITY DOMAIN
See attached QMRF.

6. ADEQUACY OF THE RESULT
The result is to be used as part of a weight-of-evidence approach to fulfil the skin sensitisation endpoint with other in vitro and in vivo tests

Reason / purpose for cross-reference:

other: Weight of evidence

Principles of method if other than guideline:

- Software tool(s) used including version: QSAR
- Model(s) used: DEREK NEXUS Version 6.1.0
- Model description: see field, 'Attached justification''
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

no

Justification for non-LLNA method:

Not applicable

Remarks on result:

no indication of skin sensitisation

Remarks:

The QSAR prediction program indicates that there no structural alerts for skin sensitisation.

Outcome of the prediction model:

negative [in vitro/in chemico]

The query structure does not match any structural alerts or examples of skin sensitisation in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitiser.

Conclusions:

The substance is not predicted to be a skin sensitiser using the DEREK NEXUS QSRA program.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Conclusion based on a weight-of-evidence approach

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the in vitro/vivo studies conducted on skin, results of these studies indicate that the substance did not meet the test criteria to be classified as a skin sensitiser.  In addition, the chemical structure of the substance does not indicate that the substance would be a skin sensitiser.  Based on a weight-of-evidence approach plus the fact that the substance did not have a SI value >3 in the LLNA-ELISA type assay support non-classification.  The substance does not fulfil the criteria for classification as a skin sensitiser under the CLP regulation (EC1272/2008, as amended).